Method for generating hypermutable organisms

ABSTRACT

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. The enhanced rate of mutation can be further augmented using mutagens. Moreover, the hypermutability of mismatch repair deficient cells can be remedied to stabilize cells or mammals with useful mutations.

This application is a divisional application of U.S. Ser. No.09/853,646, filed May 14, 2001, now U.S. Pat. No. 6,825,038, whichclaims the benefit of 60/203,905 filed May 12, 2000 and 60/204,769 filedMay 17, 2000, the disclosures of which are explicitly incorporatedherein.

TECHNICAL FIELD OF THE INVENTION

The invention is related to the area of mismatch repair genes. Inparticular it is related to the field of mutagenesis.

BACKGROUND OF THE INVENTION

Within the past four years, the genetic cause of the HereditaryNonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynchsyndrome II, has been ascertained for the majority of kindreds affectedwith the disease (13). The molecular basis of HNPCC involves geneticinstability resulting from defective mismatch repair (MMR). Many geneshave been identified in rodents and humans that encode for proteins thatappear to participate in the MMR process, including the mutS homologsGTBP, hMSH2, and hMSH3 and the mutL homologs hMLH1, hPMS1, and hPMS2 (2,7, 11, 17, 20, 21, 22, 24). Germ line mutations in four of these genes(hMSH2, hMLH1, hPMS1, and hPMS2) have been identified in HNPCC kindreds(2, 11, 13, 17, 24). Though the mutator defect that arises from the MMRdeficiency can affect any DNA sequence, microsattelite sequences areparticularly sensitive to MMR abnormalities (14). Microsatteliteinstability is therefore a useful indicator of defective MMR. Inaddition to its occurrence in virtually all tumors arising in HNPCCpatients, Microsattelite instability is found in a small fraction ofsporadic tumors with distinctive molecular and phenotypic properties(27).

HNPCC is inherited in an autosomal dominant fashion, so that the normalcells of affected family members contain one mutant allele of therelevant MMR gene (inherited from an affected parent) and one wildtypeallele (inherited from the unaffected parent). During the early stagesof tumor development, however, the wildtype allele is inactivatedthrough a somatic mutation, leaving the cell with no functional MMR geneand resulting in a profound defect in MMR activity. Because a somaticmutation in addition to a germline mutation is required to generatedefective MMR in the tumor cells, this mechanism is generally referredto as one involving two hits, analogous to the biallelic inactivation oftumor suppressor genes that initiate other hereditary cancers (11, 13,25). In line with this two hit mechanism, the non-neoplastic cells ofHNPCC patients generally retain near normal levels of MMR activity dueto the presence of the wildtype allele.

A wide range of organisms with defective MMR have been found to havewidespread genetic mutations throughout their genome. In all cases,these organisms have germline mutations within both copies of aparticular MMR gene. Recently, work done by Nicolaides et al have shownthat a decrease in MMR can be achieved within cells from higher orderorganisms by introducing a dominant negative allele of a MMR gene. Thesedata suggest that the use of such an approach can generate geneticallyaltered organisms to produce new output traits. There is a need in theart for additional methods with which to generate genetic diversity.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forrendering cells hypermutable.

It is another object of the present invention to provide geneticallyaltered cell lines.

It is another object of the present invention to provide phenotypicallyaltered cell lines.

It is yet another object of the present invention to provide a method toproduce an enhanced rate of genetic hypermutation in a cell.

It is a further object of the invention to provide a method of mutatinga gene(s) of interest in a cell.

It is a further object of the invention to claim composition of matterfor a genetically altered bacterial purine phosphorlyase.

It is a further object of the invention to claim composition of matterfor a genetically altered bacterial purine phosphorlyase as a diagnostictool for monitoring mismatch repair deficiency of a eucaryotic cell.

It is a further object of the invention to claim composition of matterfor a generating genetically altered genes by incorporating apolymononucleotide tract to measure for altered mismatch repair ineucaryotic cells.

Yet another object of the invention is to provide a method of creatingcells with new phenotypes.

Yet another object of the invention is to provide a method of creatingcells with new phenotypes and a stable genome.

Yet another object of the invention is to provide a method of regulatingthe genetic stability of a cell or organism's genome.

It is a further object of the invention to generate hypermutable celllines using inducible vectors containing dominant negative mismatchrepair gene mutants.

It is a further object of the invention to screen for hypermutable celllines containing inducible vectors with dominant negative mismatchrepair gene mutants under induced gene expression conditions.

It is a further object of the invention to screen for hypermutable celllines containing inducible vectors with dominant negative mismatchrepair gene mutants under induced gene expression conditions for alteredgene structure and/or new phenotypes.

It is a further object of the invention to turn off expression of adominant negative MMR gene in cells containing structurally alteredtarget genes and/or new phenotypes to restore genomic stability.

It is a further object of the invention to screen hypermutable celllines containing an inducible vector comprising a dominant negativemismatch repair gene mutant under inducing conditions in the presence ofchemical mutagens or ionizing radiation for structurally altered targetgenes and/or new phenotypes. Cells containing altered gene structureand/or new phenotype are then removed from inducer molecule and geneticstability is restored.

These and other objects of the invention are provided by one or more ofthe embodiments described below. In one embodiment of the invention, amethod for making a hypermutable cell is provided. A polynucleotideencoding a dominant negative allele of a mismatch repair gene isintroduced into a cell. The cell becomes hypermutable as a result of theintroduction of the gene.

In another embodiment of the invention, an isolated hypermutable cellwill be provided. The cell comprises a dominant negative allele of amismatch repair gene. The cell is exposed to DNA akylating agents. Thecell exhibits an enhanced rate of hypermutation.

In another embodiment of the invention, a method is provided forintroducing a mutation into a gene of interest. A polynucleotideencoding a dominant negative allele of a mismatch repair gene isintroduced into a cell. The cell becomes hypermutable as a result of theintroduction of the gene. The cell further comprises a gene of interest.The cell is grown. The cell is tested to determine whether the gene ofinterest harbors a mutation.

In another embodiment of the invention, a method is provided forinserting a polymononucleotide tract in a gene to measure for mismatchrepair activity of a eucaryotic cell. A polynucleotide tract is insertedout-of-frame into the coding region of a gene or a cDNA. The gene isintroduced into a cell. The polymononucleotide tract is altered bymismatch repair deficiency. An in-frame altered gene is produced.

In another embodiment of the invention, a method is provided forproducing new phenotypes of a cell. A polynucleotide encoding a dominantnegative allele of a mismatch repair gene is introduced into a cell. Thecell becomes hypermutable as a result of the introduction of the gene.The cell is grown. The cell is tested for the expression of newphenotypes. Another embodiment of the invention is the use of cellscontaining an inducible vector consisting of a dominant negativemismatch repair gene mutants under inducing conditions in the presenceof chemical mutagens or ionizing radiation for altered target genesand/or new phenotypes. Cells containing altered gene structure and/ornew phenotype are then removed from inducer molecule and geneticstability is restored. The cells are now used for commercial propertiessuch as but not limited to recombinant manufacturing and/or genediscovery.

Another embodiment of the invention is the use of MMR defective cellscontaining a gene of interest in the presence of chemical mutagens orionizing radiation for altered target genes and/or new phenotypes. Cellscontaining altered gene structure and/or new phenotype are then stablytransduced with a wildtype MMR complementing gene and genetic stabilityis restored. The cells are now used for recombinant manufacturing orgene discovery.

In another embodiment of the invention, a method is provided forrestoring genetic stability in a cell with defective mismatch repairgene. The activity of the mismatch repair process is restored and itsgenome is stable.

In another embodiment of the invention, a method is provided forrestoring genetic stability in a cell with defective mismatch repairactivity and a newly selected phenotype. The MMR deficiency can occurthrough the inactivation of endogenous MMR genes via genomic mutationsor through the introduction of an eucaryotic expression vector producinga dominant negative MMR gene allele. In the case of cells lackingendogenous MMR due to a defect in an endogenous MMR gene, the cell isselected for a new phenotype or altered gene, RNA, or polypeptide. Thecell becomes genetically stable through the introduction of a normalfunctioning MMR gene that complements the genomic defect of the hostcell. This complementation group can include the use of any gene knownto participate in mismatch repair deficiency. In the case were theexpression of the dominant negative mismatch repair gene is used toinduce DNA hypermutability, the dominant negative MMR gene expressionwill be suppressed by removal of the inducer molecule or by knocking outthe expression of the dominant negative gene allele using standard geneknockout technology used by those skilled in the art (Waldman, T.,et.al. Cancer Res 55:5187-5190, 1995). In any case, the cell restoresits genetic stability and the new phenotype is stable.

These and other embodiments of the invention provide the art withmethods that can generate enhanced mutability in organisms, cells andanimals as well as providing genetically altered stable organisms cellsand animals harboring potentially useful genome alterations.

The use of a dominant negative MMR gene allele is important ingenerating global mutations throughout the genome of a host organism ina regulated fashion. While the use of dominant negative alleles havebeen previously demonstrated to be capable of inducing globalmutagenesis in a wide range of hosts (bacteria, yeast, mammals, plants)the use of inducible vectors to turn the dominant negative MMR genemutant on to generate genome-wide mutation followed by selection for newbiochemical output traits (e.g., resistance to chemical mutagens) andturning off of the dominant negative MMR gene allele to restore geneticstability is a new aspect of the invention. This method is now suitablefor generating genetically diverse prokaryotic, eucaryotic and mammaliancells that can be screened for genetic mutations in genes involved innew phenotypes. In addition, this application teaches of the use ofintroducing dominant negative MMR alleles under control of inducibleexpression elements into MMR proficient cells. Stable or transientlytransduced cells are then exposed to inducer molecule resulting inexpression of the dominant negative MMR gene. Expression of the dominantnegative product interferes with the endogenous MMR machinery, therebycausing genetic instability that leads to genetically diverse sublines.These cells are then put under specific selective assays and screenedfor new phenotypes and/or altered gene structures. After theestablishment of sublines containing altered target genes and/or newphenotypes, cells are then rendered genetically stable by removal of theinducer molecule and a stable cell line is now produced that contains analtered gene and/or exhibits a new phenotype. This cell line can be usedfor gene discovery, drug target discovery, recombinant gene mutagenesis,and/or recombinant protein production.

It is well established that MMR deficient organisms are more tolerant toDNA damaging agents such as alkylating agents or ionizing radiationthereby leading to enhanced levels of genome-wide or locus-specificmutagenesis. Here we teach the use of exposing cell lines expressingdominant negative MMR under control of an inducible expression elementto DNA damaging agents that can lead to enhanced genome widemutagenesis. Cell lines are then screened for mutations in target genesor screened for novel phenotypes. Sublines with altered genes orphenotypes are then removed from inducer agent to “turn off” thedominant negative MMR gene allele to restore genetic stability. Thiscell line can be used for gene discovery, drug target discovery,recombinant gene mutagenesis, and/or recombinant protein production.

Finally, the use of mammalian cell lines that are naturally defectivefor MMR can be used to introduce a plasmid containing a gene ofinterest. The gene can be introduced and expressed transiently orstably. The cell now grows and the structure and/or function of theintroduced gene is screened to identify those with structural and/orfunctional alterations. To enhance mutation rate, cells can be furtherexposed to DNA damaging agents such as but not limited to alkylatingchemical mutagens or ionizing radiation to produce enhanced genome widemutation rate in the host. Once a cell line(s) containing mutationswithin the gene of interest are generated, the cell is stably transducedwith a gene that complements for the endogenous MMR defect. The cellline is now genetically stable and the cell line is suitable forproducing altered gene products for gene discovery, recombinant genemutagenesis, and/or recombinant protein production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Diagrams of pCAR reporters.

FIG. 2A-2C. SH cells cotransfected with pCAR reporters and PMS2expression vectors after 17 days of drug selection. (FIG. 2A) Westernblots of lysates from untransfected SH cells (lane 1) or SH cellstransfected with PMS2NOT (lane 2) or PMS2WT (lane 3). The arrowindicates the 110 kD protein expected for hPMS2. (FIG. 2B) Western blotsof lysates from untransfected SH cells (lane 1) or SH cells transfectedwith PMS2NOT (lane 2) or PMS2134 (lane 3). The arrow indicates the 14kD-protein expected for hPMS-134. Both A and B were probed with anantibody generated against the N terminus of hPMS2. The upperpolypeptides in A and the lower polypeptides in B representcrossreactive hamster proteins. (FIG. 2C) β-galactosidase activity inlysates derived from SH cells cotransfected with PMS2NOT (lane 1),PMS2WT (lane 2), or PMS2134 (lane 3) plus reporter plasmid. Relativeβ-galactosidase activities are defined as the ratio of β-galactosidaseactivity in cells transfected with pCAROF compared to that in cellstransfected with pCARIF; this normalization controlled for transfectionefficiency and controlled for β-galactosidase activity in the cellsexpressing the various PMS2 effector genes.

FIGS. 3A and 3B. In situ β-galactosidase activity of pooled clones of SHcells stably transduced with the PMS2WT (FIG. 3A), or PMS2134 (FIG. 3B)expression vectors, then retransfected with pCAROF reporter. After 17days of drug selection, the colonies were pooled, cultured, and stainedfor β-galactosidase activity. A pooled culture of PMS2134 transduced SHcells expressing β-galactosidase from pCAROF is visible in FIG. 3B. Eachof the fields illustrated is representative of that found in triplicateexperiments.

FIG. 4. Generation of inducible mammalian expression vectors containingdominant negative mismatch repair gene alleles. The PMS134 cDNA with orwithout a V5 epitope at the C-terminus was cloned into theecdysone-steroid regulated pIND mammalian expression vector. The PMS134cDNA was cloned into the unique BamHI site of the vector in the senseorientation to the Heat shock Minimal Promoter. The resultant vectorsare referred to as pINDPMS134V5 or pINDPMS134, respectively. The pINDvector contains that neomycin resistance gene as selectable marker.

FIG. 5. Generation of altered gene sequences upon induction of PMS134.Cells containing pIND empty vector or pINDPMS134 were transfected withthe pCAR-OF plasmid containing the β-galactosidase reporter plasmid witha polyCA repeat in the N-terminus of the β-gal gene, which disrupts theopen reading frame to produce a frameshift. The plasmid also containsthe hygromycin resistance gene to select for stable lines. Cells thatwere G418/hygromycin resistant were expanded and grown for 10 days withor without 1 μM ecdysone. At day 14, cells were stained in situ for focithat produced functional β-gal. As shown, a significant number(25/field) of β-gal positive foci were observed in cells grown in thepresence of the steroid inducer while little were observed in culturesgrown without the inducer molecule.

FIG. 6. Re-establishment of genetically stable cells after selection. Todetermine if clones were genetically stable after removal of chemicalinducer (and subsequent shut down of dominant negative MMR allele),pINDPMS134/pCAR-OF clones were isolated and tested for functional β-galactivity. Clones with β-gal expression were plated in 96 well plates atlimiting dilution yielding roughly 45 well with clones per dish. Cloneswere again grown 14 generations (1 generation/day) with or withoutecdysone and stained for β-gal activity in situ. As shown, a significantnumber of β-gal positive clones were observed in cells grown in theabsence of the steroid inducer (42/45 wells were positive for β-gal)while a larger number of clones lost β-gal activity under constantinducer exposure (18/45 wells were positive for β-gal). These datademonstrate the ability to restore genetic stability in clones that havebeen genetically altered in vivo via blockade of MMR.

FIG. 7. Diagram of the genetically altered purine phosphorlyase (PNP)gene with an out-of-frame poly A tract inserted in the N-terminus(referred to as polyPNP) (SEQ ID NO: 3). This gene encodes for anon-functional PNP gene. When the polyA tract is randomly altered by adefective MMR, the tract is shifted and allows for the production of afunctional PNP gene. PNP can convert the non-toxic prodrug9-(β-D-2-deoxyerythropentofuranosyl)—6-methyl-purine (referred to asMPD) to the toxic 6-methyl purine analog (referred to as (MP). Theconstruct has a hemaglutinin (HA) tag at the C-terminus for western blotanalysis. A control construct with an in-frame polyA tract encoding afull-length polypeptide (SEQ ID NO: 4) is shown on the bottom.

FIGS. 8A and 8B. Toxicity assay of MMR defective or proficient cellsexpressing polyPNP with or without exposure to MPD. (FIG. 8A) Graphshows that in MMR-defective cells expressing the polyPNP gene, cells arekilled in the presence of the MPD prodrug in contrast to MMR-proficientcells. (FIG. 8B) Western blot that shows production of apolyPNP-HA-tagged polypeptide in the MMR defective cells in contrast toMMR-proficient cells.

FIG. 9. Toxicity assay of a MMR defective or proficient cell lineexpressing polyPNP with or without exposure to MPD. The graph shows thatin MMR-defective HCT116 cells (genetically deficient for MLH1), theintroduction of a functional MLH1 gene restores the genetic stability ofthe cell as indicated by the fact that the polyPNP gene is not convertedto an active form as seen in HCT116 cells transfected with a truncated(non-functional) MLH1 cDNA (pC9MLHstop). These data demonstrate that MMRdeficiency can be complemented with a functional MMR gene(HCT116/pC9MLH1), therefore maintaining the genomic integrity of a geneor locus that has been altered.

FIGS. 10A and 10B. Western blot of cells transfected with an MLH1expression construct and the polyPNP gene. (FIG. 10A) Cell lysates fromcells transfected with the MLHstop expression vector (lane 1) or theMLH1 vector (lane 2) were lysed and probed for MLH1 protein expressionin HCT116 cells. As shown in FIG. 10A, the cells transfected with theMLH1 full-length expression construct produced a polypeptide of theexpected molecular weight (arrow). (FIG. 10B) Cell lysates from HCT116cells transfected with the MLHstop expression vector (lane 1) or theMLH1 vector (lane 2) plus the polyPNP gene were lysed and probed forpolyPNP using an anti-HA monoclonal antibody that can detect the HA tagat the C-terminus of the PNP protein. As shown in FIG. 10B, the cellstransfected with the MLHstop expression construct produced a polypeptideof the expected molecular weight (arrow) in contrast to cellstransfected with the functional MLH1 cDNA, which restored genomicstability of the cell therefore maintaining the genomic structure of thepolyPNP gene.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered a method for developing hypermutable cellsby taking advantage of cells with mismatch repair deficiency to createaltered genes, RNAs, polypeptides and cells or whole organisms with newphenotypes. Dominant negative alleles of such genes, when introducedinto cells or transgenic animals, increase the rate of spontaneousmutations by reducing the effectiveness of DNA repair and thereby renderthe cells or animals hypermutable. Hypermutable cells or animals canthen be utilized to develop new mutations in a gene(s) to produce newoutput traits of a host cell or organism. The inventors will show thatthe use of chemical agents that cause damage to DNA can enhance the rateof hypermutability in cells expressing dominant negative mismatch repairgene alleles. The inventors also show that the selection of alteredgenes and restoration of genetic stability of a host cell or organism byrestoring MMR can lead to stable biological products consisting ofaltered genes, RNAs, or polypeptides.

Protein complexes in cells ranging from bacteria to mammalian cellscarry out the process of mismatch repair, also called mismatchproofreading. A mismatch repair gene is a gene that encodes one of theproteins of such a mismatch repair complex. Although not wanting to bebound by any particular theory of mechanism of action, a mismatch repaircomplex is believed to detect distortions of the DNA helix resultingfrom non-complementary pairing of nucleotide bases. Thenon-complementary base on the newer DNA strand is excised, and theexcised base is replaced with the appropriate base which iscomplementary to the older DNA strand. In this way, cells eliminate manymutations that occur as a result of mistakes in DNA replication.

Dominant negative alleles cause a mismatch repair defective phenotypeeven in the presence of a wild-type allele in the same cell. An exampleof a dominant negative allele of a mismatch repair gene is the humangene hPMS2-134, which carries a truncation mutation at codon 134. Themutation causes the product of this gene to abnormally terminate at theposition of the 134th amino acid, resulting in a shortened polypeptidecontaining the N-terminal 133 amino acids. Such a mutation causes anincrease in the rate of mutations which accumulate in cells after DNAreplication. Expression of a dominant negative allele of a mismatchrepair gene results in impairment of mismatch repair activity, even inthe presence of the wild-type allele. Any allele which produces sucheffect can be used in this invention.

Dominant negative alleles of a mismatch repair gene can be obtained fromthe cells of humans, animals, yeast, bacteria, or other organisms.Screening cells for defective mismatch repair activity can identify suchalleles. Cells from animals or humans with cancer can be screened fordefective mismatch repair. Cells from colon cancer patients may beparticularly useful. Genomic DNA, cDNA, or mRNA from any cell encoding amismatch repair protein can be analyzed for variations from the wildtype sequence. Dominant negative alleles of a mismatch repair gene canalso be created artificially, for example, by producing variants of thehPMS2-134 allele or other mismatch repair genes. Various techniques ofsite-directed mutagenesis can be used. The suitability of such alleles,whether natural or artificial, for use in generating hypermutable cellsor animals can be evaluated by testing the mismatch repair activitycaused by the allele in the presence of one or more wild-type alleles,to determine if it is a dominant negative allele.

A cell, an organism, or an animal into which a dominant negative alleleof a mismatch repair gene has been introduced will become hypermutable.This means that the spontaneous mutation rate of such cells or animalsis elevated compared to cells or animals without such alleles. Thedegree of elevation of the spontaneous mutation rate can be at least2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold,or 1000-fold that of the normal cell or animal.

According to one aspect of the invention, a polynucleotide encoding adominant negative form of a mismatch repair protein is introduced intoany eucaryotic cell or a transgenic animal. The gene can be any dominantnegative allele encoding a protein, which is part of a mismatch repaircomplex, for example, PMS2, PMS1, MLH1, GTBP, MSH3 or MSH2. The dominantnegative allele can be naturally occurring or made in the laboratory.The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or achemically synthesized polynucleotide. The polynucleotide can be clonedinto an expression vector containing a constitutively active promotersegment (such as but not limited to CMV, SV40, EF-1

or LTR sequences) or to inducible promoter sequences such as thetetracycline, or ecdysone/glucocorticoid inducible vectors, where theexpression of the dominant negative mismatch repair gene can beregulated. The polynucleotide can be introduced into the cell bytransfection.

Transfection is any process whereby a polynucleotide is introduced intoa cell. The process of transfection can be carried out in a livinganimal, e.g., using a vector for gene therapy, or it can be carried outin vitro, e.g., using a suspension of one or more isolated cells inculture. The cell can be any type of eucaryotic cell, including, forexample, cells isolated from humans or other primates, mammals or othervertebrates, invertebrates, and single celled organisms such as protozoaor yeast.

In general, transfection will be carried out using a suspension ofcells, or a single cell, but other methods can also be applied as longas a sufficient fraction of the treated cells or tissue incorporates thepolynucleotide so as to allow transfected cells to be grown andutilized. The protein product of the polynucleotide may be transientlyor stably expressed in the cell. Techniques for transfection are wellknown. Available techniques for introducing polynucleotides include butare not limited to electroporation, transduction, cell fusion, the useof calcium chloride, and packaging of the polynucleotide together withlipid for fusion with the cells of interest. Once a cell has beentransfected with the mismatch repair gene, the cell can be grown andreproduced in culture. If the transfection is stable, such that the geneis expressed at a consistent level for many cell generations, then acell line results.

An isolated cell is a cell obtained from a tissue of humans or animalsby mechanically separating out individual cells and transferring them toa suitable cell culture medium, either with or without pretreatment ofthe tissue with enzymes, e.g., collagenase or trypsin. Such isolatedcells are typically cultured in the absence of other types of cells.Cells selected for the introduction of a dominant negative allele of amismatch repair gene may be derived from a eucaryotic organism in theform of a primary cell culture or an immortalized cell line, or may bederived from suspensions of single-celled organisms.

A polynucleotide encoding a dominant negative form of a mismatch repairprotein can be introduced into the genome of an animal by producing atransgenic animal. The animal can be any species for which suitabletechniques are available to produce transgenic animals. For example,transgenic animals can be prepared from domestic livestock, e.g., cows,pigs, sheep, goats, horses, etc.; from animals used for the productionof recombinant proteins, e.g., cows, pigs, or goats that express arecombinant protein in their milk; or experimental animals for researchor product testing, e.g., mice, rats, hamsters, guinea pigs, rabbits,etc.

Any method for making transgenic animals known in the art can be used.According to one process of producing a transgenic animal, thepolynucleotide is injected into a fertilized egg of the animal and theinjected egg is placed into a pseudo-pregnant female. The egg developsinto a mature animal in which the polynucleotide is incorporated andexpressed. The fertilized egg is produced in vitro from the egg andsperm of donor animals of the same species as the pseudo-pregnantfemale, who is prepared by hormone treatments to receive the fertilizedegg and become pregnant. An alternative method for producing transgenicanimals involves introducing the polynucleotide into embryonic cells byinjection or transfection and reintroducing the embryonic cells into thedeveloping embryo. With this method, however, if the polynucleotide isnot incorporated into germ line cells, the gene will not be passed on tothe progeny. Therefore, a transgenic animal produced by this method mustbe evaluated to determine whether the gene is incorporated into germcells of the animal. Once transgenic animals are produced, they can begrown to reproductive age, when they can be mated to produce andmaintain a colony of transgenic animals.

Once a transfected cell line or a colony of transgenic animals has beenproduced, it can be used to generate new mutations in one or moregene(s) of interest. A gene of interest can be any gene naturallypossessed by the cell line or transgenic animal or introduced into thecell line or transgenic animal. An advantage of using such cells oranimals to induce mutations is that the cell or animal may have a widespectrum of genetic alterations that may produce commercially beneficialbiological products. Hypermutable animals can then be bred and selectedfor new desired output traits (such as milk production, pest resistance,etc.). Once a new trait is identified, the dominant negative allele canbe removed by directly knocking out the allele by technologies used bythose skilled in the art or by breeding to mates lacking the dominantnegative allele to select for offspring with a desired trait and astable genome. Another alternative is to use a CRE-LOX expressionsystem, whereby the dominant negative allele is spliced from the animalgenome once a new output trait has been established.

Another aspect of the invention is the use of cells lacking MMR (due tomutated endogenous MMR gene or genes or through the introduction of adominant negative MMR gene) and chemical mutagens to cause an enhancedrate of mutations in a host's genome. The lack of MMR activity has beenknown to make cells more resistant to the toxic effects of DNA damagingagents. This invention teaches of the use of making proficient MMRcells; mismatch repair defective via the expression of a dominantnegative MMR gene allele and then enhancing the genomic hypermutabilitywith the use of a DNA mutagen. This application also teaches us of theuse of employing cells that are naturally deficient in MMR and exposureof chemical mutagens to increase the rate of genomic alterations togenerate cells with new genetic structures and/or new phenotypes.Chemical mutagens are classifiable by chemical properties, e.g.,alkylating agents, cross-linking agents, etc. The following chemicalmutagens are useful, as are others not listed here, according to theinvention. N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU),procarbazine hydrochloride, chlorambucil, cyclophosphamide, methylmethanesulfonate (MMS), ethyl methanesulfonate (EMS), diethyl sulfate,acrylamide monomer, triethylene melamin (TEM), melphalan, nitrogenmustard, vincristine, dimethylnitrosamine,N-methyl-N′-nitro-Nitrosoguanidine (MNNG), 7,12 dimethylbenz (a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan. Ina preferred aspect of the invention, a mutagenesis technique is employedthat confers a mutation rate in the range of 1 mutation out of every 100genes; 1 mutation per 1,000 genes. The use of such combination (MMRdeficiency and chemical mutagens will allow for the generation of a widearray of genome alterations (such as but not limited to expansions ordeletions of DNA segments within the context of a gene's coding region,a gene's intronic regions, or 5′ or 3′ proximal and/or distal regions,point mutations, altered repetitive sequences) that are preferentiallyinduced by each particular agent.

Mutations can be detected by analyzing for alterations in the genotypeof the cells or animals, for example by examining the sequence ofgenomic DNA, cDNA, messenger RNA, or amino acids associated with thegene of interest. Mutations can also be detected by screening thephenotype of the gene. An altered phenotype can be detected byidentifying alterations in electrophoretic mobility, spectroscopicproperties, or other physical or structural characteristics of a proteinencoded by a mutant gene. One can also screen for altered function ofthe protein in situ, in isolated form, or in model systems. One canscreen for alteration of any property of the cell or animal associatedwith the function of the gene of interest, such as but not limited tomeasuring protein secretion, chemical-resistance, pathogen resistance,etc.

Another invention of the application is the use of inducible vectorsthat control the expression of a dominant negative and normallyfunctioning MMR gene. This application teaches of the utility of usingsuch a strategy to restore DNA stability once a host cell or organismexhibiting a new output trait, altered gene, RNA or polypeptide has beengenerated via trait selection with or without the combination ofchemical mutagens to establish a genetically stable version of this cellor organism. In the case of MMR defective cells as a result ofectopically expressing a dominant negative MMR gene allele, the MMRactivity is decreased or completely eliminated by removing the inducermolecule from the cell culture or organism's environment. In addition,the expression of a dominant negative MMR gene can be suppressed byknocking out the MMR gene allele using methods that are standard tothose skilled in the art of DNA knockout technology in germ or somaticcells (Waldman, T., et.al. Cancer Res 55:5187-5190, 1995).

Yet another invention teaches us of the use of restoring MMR activity ina MMR defective cell line such as HCT116, DLD-1, etc., whereby the cellis treated with chemical mutagens, selected for a new output trait suchas pathogen-resistance, chemical-resistance, etc. The cell is thentransfected with a copy of a wild type MMR gene that complements theendogenous MMR defect and restores DNA stability of a cell or anorganism exhibiting a new output trait, an altered gene sequence, analtered RNA expression and/or an altered protein expression.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLE 1 Use of Dominant Negative Mismatch Repair Protein to CauseHypermutability in Mismatch Repair Proficient Cells

A profound defect in MMR was found in the normal cells of two HNPCCpatients. That this defect was operative in vivo was demonstrated by thewidespread presence of microsattelite instability in nonneoplastic cellsof such patients. One of the two patients had a germline truncatingmutation of the hPMS2 gene at codon 134 (the hPMS2134 mutation), whilethe other patient had a small germline deletion within the hMLH1 gene(26). These data thus contradicted the twohit model generally believedto explain the biochemical and biological features of HNPCC patients.The basis for this MMR deficiency in the normal cells of these patientswas unclear, and several potential explanations were offered. Forexample, it was possible that the second allele of the relevant MMR genewas inactivated in the germline of these patients through anundiscovered mechanism, or that unknown mutations of other genesinvolved in the MMR process were present that cooperated with the knowngermline mutation. It is clear from knockout experiments in mice thatMMR deficiency is compatible with normal growth and development,supporting these possibilities (1,3,6). Alternatively, it was possiblethat the mutant alleles exerted a dominant negative effect, resulting inMMR deficiency even in the presence of the wildtype allele of thecorresponding MMR gene and all other genes involved in the MMR process.To distinguish between these possibilities, we expressed the truncatedpolypeptide encoded by the hPMS2134 mutation in an MMR proficient cellline and analyzed its affect on the cell's MMR activity. The resultsshowed that this mutant could indeed exert a dominant negative effect,resulting in biochemical and genetic manifestations of MMR deficiency.

The MMR proficient Syrian hamster TKts13 cell line (hereafter called SHcells) was cotransfected with various hPMS2 expression plasmids plusreporter constructs for assessing MMR activity. The hPMS2 expressionplasmids contained the normal hPMS2 gene product or the truncated hPMS2gene identified in the patient described above (PMS2WT and PMS2134,respectively, FIG. 1A). An “empty” vector devoid of hPMS2 sequences(PMS2NOT, FIG. 1A) served as an additional control. The reporterconstruct pCAROF (out of frame) contained a hygromycin resistance geneplus a β-galactosidase gene containing a 29 bp outofframe polyCA tractat the 5′ end of its coding region. The reporter construct pCARIF (inframe) was identical except that the polyCA tract was 27 bp andtherefore did not disrupt the β-galactosidase reading frame (FIG. 1B).The pCAROF reporter would not generate β-galactosidase activity unless aframerestoring mutation (i.e., insertion or deletion) arose followingtransfection.

Different transfection schemes were used to evaluate the effects of thePMS2134 mutation on SH cells. In the first scheme, the expressionvectors plus the reporters were cotransfected together. Pools containinggreater than 100 clones and individual clones were generated followingselection with hygromycin for 17 days and harvested for Western blot andβ-galactosidase assays. SH cells transduced with PMS2WT and PMS2134synthesized polypeptides of the expected size, as assessed withantihPMS2 antibodies on Western blots (FIGS. 2A and 2B). As expected,virtually no β-galactosidase activity was observed in SH cellstransfected with the pCAROF reporter plus PMS2NOT (FIG. 2C). However, SHcells transfected with PMS2134 expressed considerable β-galactosidaseactivity, significantly more than those transfected with PMS2WT (FIG.2C). These results suggested that the truncated polypeptide encoded bythe PMS2134 construct perturbs the endogenous MMR machinery, resultingin deletions or insertions that restored the reading frame. The exactnature of these presumed deletions or insertions could not be assessed,as multiple copies of the reporter constructs were transduced under ourconditions, and the wild type β-galactosidase sequence was in greatexcess over the expected mutants, precluding their demonstration bydirect sequencing.

In the second scheme, SH cells were cotransfected with each of the PMS2expression vectors plus the hygromycin resistance plasmid pLHL4.Hygromycin resistant cultures containing greater than 100 clones werepooled and expanded. These cultures were then cotransfected with pCARIFor pCAROF reporters plus a separate plasmid allowing geneticinselection. Two weeks later, the pooled cells, each containing more than100 colonies resistant to both hygromycin and geneticin, were stainedwith Xgal to assess β-galactosidase activity. As shown in FIG. 3, thecultures transfected with PMS2134 (panel B) contained many blue cells,while virtually no cells were blue in the cultures transfected withPMS2WT (panels A). In each case, transfection efficiency was controlledby parallel transfections using pCARIF, which also served as a controlfor β-galactosidase activity of cells expressing the various PMS2effector genes, which resulted in similar β-galactosidase expressionlevels in all cases (not shown). Increases in β-galactosidase activityafter PMS2134 transfection compared to PMS2WT transfection were alsoobserved when a similar experimental protocol was applied to theMMRproficient human embryonic kidney cell line 293. These cells werecotransfected with the pCAROF plus the various PMS2 effector plasmidsand selected for 17 days in hygromycin. At day 17, colonies were stainedwith Xgal to assess β-galactosidase activity and scored forβ-galactosidase expressing cells. As shown in Table 1, only those cellsexpressing the PMS2134 polypeptide expressed a detectableβ-galactosidase activity. These data demonstrate a similar dominantnegative effect of the hPMS2134 protein in both rodent and human systemsand validate the utility of the rodent system in these studies.

TABLE 1 β-galactosidase expression of 293 clones transfected with pCAROFreporter construct plus PMS2 effector plasmids. 293 cells werecotransfected with the pCAROF β-galactosidase reporter plasmid plus thePMS2NOT, WT, or -134 effector plasmids. Transfected cells were selectedin hygromycin for 17 days and stained with xgal for β-galactosidaseactivity (blue colored cells). The results below represent the mean +/standard deviation of triplicate experiments. Sample Blue colonies Whitecolonies PMS2NOT 0 +/ 0 17 +/ 2.7 PMS2WT 0 +/ 0 18 +/ 4.0 PMS2134  15 +/2.1  6 +/ 2.1

Plasmids. The fulllength wildtype hPMS2 cDNA was obtained from a humanHeLa cDNA library as described (18). An hPMS2 cDNA containing atermination codon at amino acid 134 was obtained via RTPCR from thepatient in which the mutation was discovered (9). The cDNA fragmentswere cloned into the BamHI site into the pSG5 vector, which contains anSV40 promoter followed by an SV40 polyadenylation signal (8). The pCARreporter vectors described in FIG. 1 were constructed as described inref. 21 and 25.

Cell lines and transfection. Syrian Hamster fibroblast Tkts13 and HumanHEK293 cells were obtained from ATCC and cultured as described (15).Stably transfected cell lines expressing hPMS2 were created bycotransfection of the PMS2 expression vectors and the pLHL4 plasmidencoding the hygromycin resistance gene at a ratio of 3:1 (pCAR:pLHL4)and selected with hygromycin. Stably transfected cell lines containingpCAR reporters were generated by cotransfection of pCAR vectors togetherwith either pNTK plasmid encoding the neomycin resistance plasmid orwith pLHL4. All transfections were performed using calcium phosphate aspreviously described (15).

β-galactosidase assay. Seventeen days following transfection with pCAR,β-galactosidase assays were performed using 20 g of protein in 45 mM2mercaptoethanol, 1 mM MgCl₂, 0.1 M NaPO₄ and 0.6 mg/ml Chlorophenol redβ-D-galatopyranoside (CPRG, Boehringer Mannheim). Reactions wereincubated for 1 hour, terminated by the addition of 0.5 M Na₂CO₃, andanalyzed by spectrophotometry at 576 nm (16). For in situβ-galactosidase staining, cells were fixed in 1% glutaraldehyde in PBSand incubated in 0.15 M NaCl, 1 mM MgCl₂, 3.3 mM K₄Fe(CN)₆, 3.3 mMK₃Fe(CN)₆, 0.2% XGal for 2 hours at 37° C.

Western Blot.

Western blots for PMS2 were performed as described in example 5 using apolyclonal anti-human PMS2 raised against the codons 1-20 of the humanfull-length polypeptide.

EXAMPLE 2 Dominant Negative Mismatch Repair Gene Alleles Cause a Defectin MMR Activity

The most likely explanation for the differences in β-galactosidaseactivity between PMS2WT and PMS2134 transfected cells was that thePMS2134 protein disturbed MMR activity, resulting in a higher frequencyof mutation within the pCAROF reporter and reestablishing the ORF. Todirectly test the hypothesis that MMR was altered, we employed abiochemical assay for MMR with individual clones from cells containingthe PMS2-WT, PMS2-134 or PMS2-NOT empty vectors as described inexample 1. Nuclear extracts were prepared from the clones and incubatedwith heteroduplex substrates containing either a /CA\ insertiondeletionor a G/T mismatch under conditions described previously. The /CA\ andG/T heteroduplexes were used to test repair from the 3′ and 5′directions, respectively. There was a dramatic difference between thePMS2-134 expressing clones and the other clones in these assays (Table2A). While all clones repaired substrates from the 3′ direction(/CA\heteroduplex), cells expressing the PMS2134 polypeptide had verylittle 5′ repair activity. A similar directional defect in mismatchrepair was evident with pooled clones resulting from PMS2134transfection, or when the heteroduplex contained a 24 base pair loop,examples of which are shown in Table 2B. A small decrease in MMRactivity was observed in the 3′/CA\PMS2-WT repair assays, perhaps aresult of interference in the biochemical assays by over expression ofthe PMS2 protein. No significant activity was caused by PMS2-WT in thein situ β-galactosidase assays (FIG. 3; Table 1), a result more likelyto reflect the in vivo condition.

TABLE 2 Mismatch repair activity of nuclear extracts from SH clones (A)or pooled cultures (B). The extracts were tested for MMR activity with24 fmol of heteroduplex. A. SH clones* Repaired substrate (fmol/15 min)Cell Line 3′/CA\5′G/T PMS2-NOT clone A 10.2 3.5 clone B 12.7 2.9 clone C13.5 5.5 PMS2-WT clone A 2.8 2.2 clone B 5.7 4.8 clone C 4.7 2.9PMS2-134 clone A 2.5 0.0 clone B 2.4 0.0 clone C 5.0 0.5 B. Pooledcultures Repaired substrate (fmol/15 min) Cell Line 3′G/T 5′G/T 3′/CTG\5′/CTG\ PMS2-NOT 2.07 +/− 0.09 2.37 +/− 0.37 3.45 +/− 1.35 2.77 +/− 1.37PMS2-WT 1.65 +/− 0.94 1.86 +/− 0.57 1.13 +/− 0.23 1.23 +/− 0.65 PMS2-1340.14 +/− 0.2   0.0 +/− 0.0  1.31 +/− 0.66  0.0 +/− 0.0  *These datarepresent similar results derived from greater than five independentexperiments.

Biochemical assays for mismatch repair. MMR activity in nuclear extractswas performed as described, using 24 fmol of substrate (12,25).Complementation assays were done by adding˜100 ng of purified MutL α orMutSα components to 100 μg of nuclear extract, adjusting the final KClconcentration to 100 mM (4,10,30). The substrates used in theseexperiments contain a strand break 181 nucleotides 5′ or 125 nucleotides3′ to the mismatch. Values represent experiments performed at least induplicate.

EXAMPLE 3 Use of MMR Defective Cells and Chemical Mutagens to EnhanceMutations in Genetic Loci

To enhance the rate of genetic mutations and produce cells with alteredgenes, RNA expression, or polypeptides, the use of MMR deficiency andchemical mutagens is a powerful tool to generate such diversity. Theadvantages of using MMR defective cells is that the decrease of thisactivity renders cells more resistant to the toxic effects of suchcompounds yet allows for the increase in genetic and phenotypicalterations of a host organism or cell. The following experiments areperformed to demonstrate the utility of the invention. Cells that aregenetically defective for MMR such as but not limited to HCT116, DLD-1,etc. or cells such as those described in example 1 and 2 that are madeMMR defective by ectopic expression of a dominant negative allele iscovered under this invention. Briefly, MMR proficient and deficientcells are incubated with a range (1 nm to 1 mM) of chemical mutagens for1 hour to 24 hours at 37 C. at 5% CO₂ in growth medium. After incubationis complete, chemical mutagens are washed from medium and cells aregrown in the presence of hypoxanthine, aminopterin, and thymine to scorefor HPRT mutant cells as previously described (Walker, V E et.al. MutatRes. 17:371-388, 1999.) and known to those skilled in the art. Cells areplated at 1×10⁵ cell ml in 10 cm tissue culture dishes and grown for 14days at 37 C. at 5% CO₂ in growth medium. After 14 days, the numbers ofHAT-resistant colonies are determined by counting under the microscope.A typical experiment will demonstrate that a significantly greaternumber of HAT resistant colonies (due to altered HPRT gene) are formedin chemically treated MMR defective cells than in control cells,demonstrating the ability to increase mutations within an endogenousgene of the host cell/organism. The use of MMR defective cells plusexposure to chemical or ionizing radiation can also be used to enhancegenetic mutation in vivo within target genes introduced via transfectionand screening of transient or stable cell lines. In order to demonstratethe ability of MMR deficiency plus chemical mutagens to enhance geneticmutation within a transduced target gene, we employed the use of cellsdescribed above, whereby the pCAROF vector (see EXAMPLE 1) wastransfected into HCT116 cells. Cells were selected for pCAR-OF positiveclones via hygromycin resistance. Hygromycin-resistant cells were grownto confluence and 100,000 cells were exposed to 10 μMethyl-methane-sulfonate (EMS) alkylating compound for 8 hours andreturned to growth medium. Cells were then grown overnight and thenplated at a density of 1,000 cells plate in 10 cm dishes in triplicate.Cells were grown for 10 days and scored for β-gal activity using methodsdescribed in EXAMPLE 1. The results showed that cells grown in theabsence of the compound the number of β-gal positive foci were 92+/−10per dish. In contrast, cells exposed to EMS resulted in a significantincrease in the number of β-gal positive cells (205+/−18). These datademonstrate the use of MMR defective cells plus chemical mutagens togenerate genetic mutations in target genes in vivo. This method isuseful for generating genetic diversity in target genes for commercialpurposes.

EXAMPLE 4 Restored DNA Stability of a Mismatch Repair Deficient CellExpressing a Dominant Negative MMR Gene Allele by Inducible Vector

The ability to induce DNA hypermutability using ectopic expression of adominant negative MMR gene allele has many important commercialapplications for generating eucaryotic cells with genetically diversesubtypes. The following experiments demonstrate the ability topermanently imprint a genetic change in the genome of a MMR defectivecell as described in Examples 1 and 2 by restoring its MMR proficiency.First, the PMS2-134 dominant negative allele was cloned into theeucaryotic inducible vector systems pcDNA4/TO (tetracycline-induciblevector) (Invitrogen), referred to as pcDNA4/TO/PMS134S, the pIND/V5-Hisglucocorticoid inducible vector (Invitrogen), referred to aspIND/PMS134S. Tk-ts13 or HEK293 cells were cotransfected with eachvector plus the pCAR-OF (contains hygromycin-resistance gene asselectable marker) as described below. An empty vector was used ascontrol for each combination. Transfected cells were selected for 10-14days for zeocin/hygromycin (Z/H) or neomycin/hygromycin (N/H) resistantcells. Clones were picked and expanded as individual clones or pools.Cells were expanded and plated in 6-well tissue culture plates at 1×10⁵cell/ml in growth medium (DMEM plus 10% fetal bovine serum) with orwithout inducer chemicals (1 μg/ml of tetracycline for pcDNA4/TO/PMS134Sand 1 μM ecdysone for pIND/PMS134S). Cell cultures were harvested andanalyzed for PMS2-134 induced protein expression via western (asdescribed in example 5) after 24 hours of culture at 37° C. in 5% CO₂.Western analysis of extracts of PIND/PMS134S cells revealed productionof a protein of˜17 kd when grown in the presence of ecdysone, whilethose grown without ecdysone had no detectable levels. Clones that haveinducible PMS2-134 expression were expanded and grown in the presence ofecdysone or tetracycline for 24 hours. Cells were harvested for 72 hoursto identify the kinetics of loss of PMS2-134 expression via western blotin the absence of inducer. These data demonstrated undetectable levelsof proetin ater 72 hours.

To demonstrate the ability to induce genetic instability using aninducible vector system, cells containing the pIND/PMS134S orpIND/V5-His were grown for 14 days with or without ecdysone and stainedto measure β-gal activity in situ as described (MCB paper). As shown inFIG. 5, a significant number of cellular foci stained positive blue inpIND/PMS134S cells grown in the presence of ecdysone (25 cells/field asobserved under inverted microscopic evaluation) in contrast to emptyvector controls which had no observable blue foci. In contrast, neitherthe pIND/PMS134S nor the pIND/V5-His cells grown in the absence of theinducer stained positive. These data demonstrate the ability of usingdominant negative MMR genes under control of inducible vectors togenerate genomic instability and genetic diversity in genes to producealtered biochemical functions and/or new phenotypes.

To demonstrate that suppressing PMS2-134 expression can restore MMRproficiency in these cells, the following experiment was performed.Cells were maintained in inducer medium plus Z/H or N/H for 14 days. Asubset of each clone or pool was plated into 24-well falcon dishes at5×10⁴ cell/ml. Cells were grown overnight at 37° C. in 5% CO₂. The nextday, cells were stained in vivo for β-galactosidase expression aspreviously described (Nicolaides et.al. Mol. Cell Biol. 18:1635-1641,1998). Cells that turn blue have done so because of a decrease in theirendogenous MMR activity due to the dominant negative effects of PMS2-134on the MMR machinery. These cells were subcloned by limiting dilution in96 well plates in the presence or absence of inducer molecule. Restoredgenetic stability was demonstrated in the PMS2-134 expressing cloneswhen a lower number of revertants (non-blue cells) were found in theclones where the inducer agent was removed (42 out of 45 wells incontrast to plates where clones were under constant exposure to inducer(18 out of 45 wells)). These data demonstrate the ability to regulategenomic stability and genetic evolution using regulated MMR geneexpression.

The use of chemical mutagens as described EXAMPLE 3 in combination withthe inducible MMR gene strategy described above are also taught in theapplication as a method for generating genetically diverse hostorganisms with new phenotypes and/or for stable production of alteredgene expression. To demonstrate this effect, cells containing inducibledominant negative expression are exposed to inducer molecule andsubsequently exposed to chemical mutagen or ionizing radiation. Cellsare then expanded in the presence of inducer molecule and cultures areselected for cells with new phenotypes and/or altered gene structure asdetermined by sequence analysis or biochemical activity. Cells withaltered gene or phenotype are then removed from inducer molecule andgenetic stability and phenotype are restored.

Transfections

Generation of stable HEK293 cells containing the ecdysone receptor withthe pIND/PMS134S or pcDNA/TO/PMS134S inducible vector. HEK293-ecdysoneRcells were transfected with the pIND or pcDNA/TO empty vector or thepIND/PMS134S or pcDNA/TO/PMS134S vector using Lipofectamine 2000(Gibco/BRL). Cells were selected for selectable marker resistance andclones and pools were expanded. Stable lines were then exposed to 1 μMecdysone for 48 hours and extracts were isolated and analyzed by westernblot to identify clones with induced PMS134 expression using antiserumdirected to the N-terminus of the PMS134 polypeptide as described below.

Plasmids

The PMS2-134 was cloned as a BamHI fragment from the pSG5PMS134(described in example 1) vector into the following inducible expressionvectors. The tetracycline inducible vector (pcDNA4/TO/PMS134S) containsthe zeocin selectable marker under control of the EM-7 promoter and SV40polyA sequences. The structure of the plasmid was confirmed byendonuclease restriction analysis and sequencing. The glucocorticoidinducible vector (pIND/PMS134S) contains the neomycin selectable markerunder control of the SV40 early promoter and polyA sequences. Aschematic figure of the vector is presented in FIG. 4.

Transfections

Inducible expression vectors were co-transfected into Tk-ts13 cells andHEK293 cells following the methods described above either alone or incombination with the pCAR-OF vector as described in EXAMPLE 1. Cellswere selected for zeocin/hygromycin (pcDNA4/TO/PMS134S) orneomycin/hygromycin (pIND/PMS134S) resistant clones as described (ref15, Grasso et.al. J. Biol. Chem. 273:24016-24024, 1998). Resistantclones are picked and/or pooled and expanded for protein analysis.

EXAMPLE 5 Restoration of MMR and Restoration of a Genetic Stability byExpressing a MMR Gene Complementing Gene and Establishment of a FixedGenomic Structure

The use of cells with defective MMR repair due to defects of endogenousgenes such as but not limited to the HCT116, DLD-1, and HEC-1-A cellslines (ref. 12, 25 and Kondo, et.al. J Biochem 125:818-825, 1999) can beuseful for altering the genetic structure of genes to producecommercially viable variant molecules such as novel anti-microbialagents, bioactive growth factors or hormones, altered antibodystructures, etc. The utility and value of such a cell is that once analtered gene structure has been produced, the integrity of this genealteration can be preserved in the cell's genome by making the cellgenetically stable via the introduction of a functional complementingMMR gene. This example demonstrates that the introduction of agenetically altered bacterial purine nucleotide phosphorlyase (PNP)gene, where an out-of-frame poly-A tract is inserted at the N-terminusof the gene (referred to as polyPNP), can be genetically altered in aMMR deficient cell and also be genetically stable when a MMR defectivecell is made MMR proficient by the direct expression of a complementingMMR gene. The polyPNP gene encodes for a non-functional PNP gene. Whenthe poly-A tract is randomly altered by genetic alterations due todefective MMR, the tract is randomly altered, allowing for theproduction of a functional PNP gene and polypeptide. PNP converts thenon-toxic 9-(β-D-2-deoxy-erythro-pento-furanosyl)-6-methyl-purineprodrug (referred to as MPD) substrate to the toxic 6-methyl purineanalog (referred to as MP) (Sorscher, E J, et.al. Gene Therapy1:233-238, 1994). The polyPNP gene was engineered to contain ahemaglutinin epitope tag at the C-terminus to facilitate the detectionof the encoded polypeptide via western blot analysis using an anti-HAantibody. The polyPNP gene was cloned into the pCEP4 expression vector,which has a hygromycin resistance (Hyg) gene for selection. Theschematic diagram showing this gene is given in FIG. 7. A homologousgene called PNP was also made in which an in-frame polyA tract is clonedinto the N-terminus of the gene as a positive control for PNP activity.Briefly, the MMR defective HCT116 cell line and the MMR proficientHEK293 cell line were transfected with the polyPNP, the PNP expressionvector, or an empty pCEP4 vector. Cells were then selected for Hygresistance and clones were isolated. Expanded cells were grown in thepresence of increasing amounts of MPD (0, 1, 10, 50, 100, 300 μM) for 10days. After treatment period, cells were counted by hemocytometer andtrypan blue exclusion. As shown in FIG. 8A, a 20% and 30% decrease incell numbers were observed when HCT116/polyPNP cells were cultured inthe presence of 100, μM or 300 μM MPD, respectively. In contrast nodecrease in cell growth was observed with the MMR proficientHEK293/polyPNP cells even at the highest concentration (300 μM) of MPDused. For both cell lines, the expression of PNP resulted in 100% growthsuppression when cells were grown in the presence of 50-300 μM MPD,demonstrating the toxic effects of the converted MP on both cell lines.Western blot confirmed that a polypeptide containing the HA epitope wasindeed produced in the HCT116/polyPNP cells, thus demonstrating thatthat the polyPNP gene structure was altered to produce a functional andfull-length PNP enzyme (FIG. 8B).

The restoration of genetic stability and the subsequent imprint of analtered gene locus or loci is an important invention of this applicationfor producing viable biological products, whereby altered biomolecules,cells or whole organisms with desired altered output traits are madegenetically stable for long term use. To generate stable MMR defectivecell lines that has or has not been exposed to chemical mutagens andselected for desired genetic changes, the introduction of acomplementing MMR gene that can substitute for the mutated endogenousMMR gene locus is taught in this application. This is demonstrated bythe example using HCT116 cells, which are genetically deficient for thehuman MutL homolog MLH1 (12, 24, 25). In this example, a mammalianexpression vector is used that encodes for the functional MLH1polypeptide (pC9MLH1) or an expression vector that encodes for a MLH1cDNA with a premature stop codon (pC9MLHstop). These expression vectorscontain a neomycin (neo) resistance gene that allows for selection ofcells containing this vector. To demonstrate the ability ofcomplementing MMR activity in an otherwise MMR defective cell and topermanently imprint the altered structure(s) of a gene locus, thepolyPNP and pC9MLH constructs were cotransfected into HCT116 cells.Cells were selected for 10 days in neo and Hyg and resistant clones wereisolated and expanded. Cells were then cultured in the presence of MPDand counted for growth after 10 days. As demonstrated in FIG. 9, cellstransfected with the MLH1 wild type cDNA expressed MLH1 as determined bywestern, in contrast to cells transfected with MLHstop. In addition,when cells were grown in the presence of 300 μM MPD, those cellsexpressing MLH1 showed a 2% decrease in total cell growth as compared tocells grown in medium alone, while cells transfected with the MLHstop orempty vector and polyPNP had a 35% reduction in cell growth incomparison to cells grown in medium alone. These data demonstrate thatcomplementing the MMR defect with an ectopically expressed wild type MMRgene or cDNA can establish genomic stability of a MMR defective cellline and establish long term stable lines that have been selected for toproduce new output traits and/or modified genomic or polypeptidestructures, such as biologically active or inactive PNP.

Plasmids.

The fulllength wildtype hMLH1 cDNA was obtained from a human Hela cDNAlibrary as described (18). A MLH1 cDNA containing a termination codonwas obtained via RTPCR from the patient in which the mutation wasdiscovered (24). The cDNA fragments were cloned into the XhoI site ofthe pCEP9 vector (Invitrogen), which contains CMV promoter followed byan SV40 polyadenylation signal (8) and a gene, which encodes forneomycin resistance. The pC9MLH1 vector produces the full-lengthfunction MLH1 protein, while the pC9MLH1 stop produces thenon-functional truncated MLH1 polypeptide. The polyPNP and PNP vectorsare described in FIG. 4. The polyPNP contains a 21 base out-of-framepolyA tract inserted after codon 2 of the bacterial PNP gene whichresults in a truncated polypeptide (Sorscher, E J, et.al. Gene Therapy1:233-238, 1994). The polyPNP contains a 20 base in-frame polyA tractinserted after codon 2 of the bacterial PNP gene which results in afull-length functionally active PNP protein. Both the polyPNP and PNPgene have a hemaglutinin (HA) epitope fused in-frame at the C-terminusfollowed by a termination codon. The polyPNP and the PNP gene wasconstructed by polymerase chain reaction using a sense primer:5′-ccaagcttagaccaccatggcaaaaaaaaaaaaaaaaaaaaatcgctaccccacacattaatgc-3′(SEQ ID NO: 1), where the polyA tract is underlined while the primer forPNP contains 1 less A in the polyA tract. The antisense primer for bothconstructs is5′-ataagaatgcggccgctatccttagctagcgtaatctggaacatcgtaagcgtaatctggaacatcgtactctttatcgcccagcag-3′(SEQ ID NO: 2). DH5α bacterial DNA was used a template foramplification. The modified PNP gene was produced by amplification USING95° C. FOR 30 SEC, 54° C. FOR 1 MINUTE, 72° C. for 1 min for 25 cyclesin buffers as previously described (19). Amplified genomic inserts werecloned into T-tailed vectors (TA cloning, Invitrogen) and recombinantclones were sequenced to identify vectors with correct nucleotidesequences. PNP fragments were then subcloned into the KpnI-XhoI sites ofthe pCEP4 vector (Invitrogen) using sites from the TA cloning vectorpolylinker. Recombinant PNP expression vectors were sequenced to ensuresequence authenticity using internal primer sequences.

Cell Lines and Transfection

Human HCT116 and HEK293 cells were obtained from ATCC and cultured assuggested by the vendor in RPMI plus 10% fetal bovine serum. Cells weretransfected with PNP and/or MLH1 expression vectors using liposomesfollowing the manufacturer's protocol (Gibco/BRL). Stably transfectedcell lines were generated that express empty vector, PNP or polyPNP bytransfection followed by hygromycin selection. For complementationexperiments, HCT116 cells were transfected with PNP/MLH1, PNP/MLH1 stop,polyPNP/MLH1 or polyPNP/MLH1 stop at a 1:1 ratio using 5 μg of eachplasmid and cells were selected for hygromycin and neomycin resistance.After 10 days, drug-resistant colonies were observed and picked foranalysis.

MPD Killing Assay

For MPD killing assay, cells were plated at 2×10⁴ cell/ml and 1 mlaliquots were plated in 24-well costar tissue culture dishes. Forkilling assays, cells were plated in 0, 1, 10, 50, 100, and 300 μM MPDin triplicate. Cells were grown for 10 days trypsinized and counted onhemocytometer using trypan blue exclusion. Data are presented as amean+/−SD for each study.

Western Blot

After counting equal cell numbers from each 0 μM MPD treated cell waslysed directly in sample buffer (60 mM Tris, pH 6.8, 2% SDS, 10%glycerol, 0.1 M 2-mercaptoethanol, 0.001% bromophenol blue) and boiledfor 5 minutes. Protein lysates were separated by electrophoresis on 18%Tris-glycine gels (Novex). Gels were electroblotted onto Inmobilon-P(Millipore) in 48 mM Tris, 40 mM glycine, 0.0375% SDS, 20% methanol andblocked at room temperature for 1 hour in Tris-buffered saline plus0.05% Tween-20 and 5% condensed milk. Filters were probed withmonoclonal antibodies (αMLH14) generated against human MLH1 orHemaglutinin (HA) (Boehringer Manheim) and a horseradish peroxidaseconjugated rabbit anti-mouse secondary antibody, using chemiluminescencefor detection (Pierce). Mouse IgG was used as control for allexperiments to assess for non-specific antibody interactions of theprimary antibody and ensure that the antiserum used were detectingexpected proteins.

REFERENCES

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1. A method for generating a mutation in a gene of interest, comprising the steps of: growing in vitro under inducing conditions at least one mammalian cell comprising (a) a gene of interest and (b) a dominant negative allele of a mismatch repair gene under control of an inducible regulatory element, wherein the dominant negative allele of a mismatch repair gene encodes a polypeptide comprising the N-terminal 133 amino acids of PMS2; contacting the at least one cell with a mutagen; and testing the at least one cell to determine whether the gene of interest harbors a mutation that results in a detectable phenotype.
 2. The method of claim 1 further comprising the step of: decreasing expression of the dominant negative allele in the selected one or more cells by culturing in non-inducing conditions.
 3. The method of claim 1 wherein expression of the dominant negative allele is decreased by site directed mutagenesis of the dominant negative allele.
 4. A method for generating a mutation in a gene of interest comprising the steps of: treating at least one cell in vitro with a mutagen, said at least one cell comprising (a) a gene of interest and (b) a dominant negative allele of a mismatch repair gene operably linked to a promoter, wherein the dominant negative allele of a mismatch repair gene encodes a polypeptide comprising the N-terminal 133 amino acids of PMS2; and testing the at least one cell to determine whether the gene of interest harbors a mutation that results in a detectable phenotype.
 5. The method of claim 4 further comprising the step of: introducing a complementing mismatch repair gene into the one or more selected cells whereby genetic stability is restored.
 6. The method of claim 5 where the complementing mismatch repair gene is constitutively active in the one or more selected cells.
 7. The method of claim 5 wherein the complementing mismatch repair gene is inducibly regulated.
 8. The method of claim 5 wherein the complementing mismatch repair gene is in PMS2.
 9. The method of claim 5 wherein the complementing mismatch repair gene is introduced into the one or more selected cells by cell-cell fusion with a mismatch repair proficient cell.
 10. The method of claim 4 wherein the testing comprises analyzing a nucleotide sequence of the gene of interest.
 11. The method of claim 4 wherein the testing comprises analyzing mRNA transcribed from the gene of interest.
 12. The method of claim 4 wherein the testing comprises analyzing an amino acid encoded by the gene of interest.
 13. The method of claim 4 wherein the testing comprises analyzing the phenotype of the cell.
 14. The method of claim 1 wherein the testing comprises analyzing a nucleotide sequence of the gene of interest.
 15. The method of claim 1 wherein the testing comprises analyzing mRNA transcribed from the gene of interest.
 16. The method of claim 1 wherein the testing comprises analyzing an amino acid encoded by the gene of interest.
 17. The method of claim 1 wherein the testing comprises analyzing the phenotype of the cell. 